9 CHAPTER 9 196 excluded in case a pathogenic mutation was present in one of the three HM genes (CACNA1A, ATP1A2 and SCN1A) or HM-related genes with mutations confirmed by Sanger sequencing.9, 31 Australian cohort. The Australian cohort was selected out of over 300 patients that had been referred to the Genomics Research Centre (GRC) Diagnostic Clinic for genetic diagnostic testing after a suspected diagnosis of HM from the referring neurologist. From this cohort, a subset of 184 (122 females and 62 males) unrelated individuals tested negative for known HM gene mutations (CACNA1A, ATP1A2, and SCN1A) and HM-related genes.9, 31 All cases consented to genetic testing with their doctors, as required under current regulations. Positive family history was reported for 25% of the cases, 5% were reported as SHM, while family information was not available for the remainder of cases. DNA was extracted from blood samples using QIAGEN QIAamp DNA Mini Kit as per the manufacturers’ instructions. Next generation sequencing (NGS) libraries for WES were constructed using the Ion AmpliSeqTM Exome RDY library kits (ThermoFisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. The Ion Chef was used to load sample libraries (barcoded fragments of 200 bp). WES was performed in the Genomics Research Centre (GRC), Australia via the Ion Proton and GeneStudio S5 plus (ThermoFisher Scientific) instruments using default settings for Ion AmpliSeq Exome RDY Kit 4x2 (ThermoFisher Scientific). The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Human Research Ethics Committee of the Queensland University of Technology (approval number: 1800000611). Dutch cohort. The cohort consisted of 32 patients (22 females and 10 males) with FHM/SHM according to ICHD-3 criteria.32 Patients were selected from the Leiden Headache Centre at the Leiden University Medical Centre (LUMC), and contained patients (i) seen in person by experienced headache clinicians or research physicians or (ii) referred from elsewhere for clinical genetic research with records being evaluated and clinical diagnosis confirmed by GMT, NP and IdB.4 All patients were from different families and did not have a known pathogenic mutation in one of the three HM genes.The study was approved by the Medical Ethics Committee of LUMC and all participants provided informed consent. Genomic DNA was extracted from peripheral blood leukocytes according to standard salting out protocol.33 WES was performed using in-house sequencing facility (Leiden Genome Technology Centre; URL: lgtc.nl) or outsourced to the Beijing Genomics Institute sequencing facility (URL: bgi.com). In brief, for the LGCT, coding sequences in the DNA were enriched using the SureSelect Human All Exon 50 Mb kit (Agilent Technologies, Santa Clara, CA, USA). Following sequence capture and amplification, fragments were sequenced using the Illumina HiSeq2000 platform (San Diego, CA, USA). Controls. As a control dataset, we used summary statistics from gnomAD (see below in the paragraph on TRAPD methods). The gnomAD database was chosen as it consists of a large
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